Park, Y.-N. | Search bioRxiv

bioRxiv2025

Interdomain disulfide bonds of rabbit kappa light chain allotypes influence mouse-rabbit chimeric antibody performance

Chimeric monoclonal antibodies have emerged as powerful tools to extend the capabilities of traditional monoclonal antibodies. These antibodies are made by replacing the variable region of a monoclonal antibody with the variable region of another antibody, typically from a different species, enabling use in a wider range of applications. Although theoretically compatible, interspecies differences in antibody structure can complicate chimeric antibody design and performance. In this study, we evaluated the impact of rabbit light chain allotype on the expression and function of chimeric antibodies containing mouse light chain variable regions. We found that constructs using the rabbit kappa 1 b4 (K1-b4) allotype frequently exhibited poor recombinant expression and, in some cases, lost antigen recognition. Structural analysis implicated disruption of an intrachain, interdomain disulfide bond as a contributing factor. Restoration of key residues predicted to re-establish this bond partially rescued both expression and activity. Additionally, chimeric antibodies incorporating the rabbit kappa 1 b9 (K1-b9) allotype, which contains a disulfide bond not disrupted by mouse variable region sequences, consistently maintained robust production and antigen-binding activity across multiple applications, including immunoblotting, immunoprecipitation, and immunostaining, for several test antibodies. Our findings underscore the importance of light chain scaffold selection in recombinant antibody engineering and provide practical guidance for optimizing chimeric antibody design to preserve both expression and function.

molecular biology↗

bioRxiv2025

Novel monoclonal antibodies against the C-terminal HEAT domain of Huntingtin

BACKGROUNDReliable detection of huntingtin (HTT) is essential for understanding Huntingtons disease (HD) biology and evaluating therapeutic strategies. However, high-quality monoclonal antibodies (mAbs) against the HTT C-terminal domain remain limited. OBJECTIVEWe sought to generate and validate novel monoclonal antibodies targeting the HTT C-terminal HEAT-containing domain to better detect HTT independently of potential effects of polyglutamine length that can impact some N-terminally targeted antibodies. METHODSWe immunized mice with a highly purified, well-characterized recombinant protein corresponding to the HTT C-terminal domain. We generated monoclonal antibody-producing hybridoma cell lines and characterized the antibodies using parental and HTT-knockout cell lines in common immuno-applications. RESULTSThree novel, independent hybridoma lines producing anti-HTT monoclonal antibodies were derived. Using CRISPR-edited HTT knockout cell lines we identified one clone, anti-HTT [2F8], that was specific and effective across Western blot, immunofluorescence, and ELISA assays. All antibodies bound full-length HTT irrespective of HAP40 interaction or polyQ length and showed no cross-reactivity to the N-terminal HEAT domain. CONCLUSIONSThese C-terminal HTT mAbs are thus valuable additional tools for studying endogenous HTT function in both normal and disease contexts.

neuroscience↗